The Journal of Neuroscience, February 28, 2018 • 38(9):2341–2358 • 2341 Neurobiology of Disease Progranulin Gene Therapy Improves Lysosomal Dysfunction and Microglial Pathology Associated with Frontotemporal Dementia and Neuronal Ceroid Lipofuscinosis X Andrew E. Arrant, Vincent C. Onyilo, Daniel E. Unger, and XErik D. Roberson Center for Neurodegeneration and Experimental Therapeutics, Alzheimer’s Disease Center, Evelyn F. McKnight Brain Institute, Departments of Neurology and Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294 Loss-of-function mutations in progranulin, a lysosomal glycoprotein, cause neurodegenerative disease. Progranulin haploinsufficiency causes frontotemporal dementia (FTD) and complete progranulin deficiency causes CLN11 neuronal ceroid lipofuscinosis (NCL). Pro- granulin replacement is a rational therapeutic strategy for these disorders, but there are critical unresolved mechanistic questions about a progranulin gene therapy approach, including its potential to reverse existing pathology. Here, we address these issues using an AAV vector (AAV-Grn) to deliver progranulin in Grn Ϫ / Ϫ mice (both male and female), which model aspects of NCL and FTD pathology, developing lysosomal dysfunction, lipofuscinosis, and microgliosis. We first tested whether AAV-Grn could improve preexisting pathol- ogy. Even with treatment after onset of pathology, AAV-Grn reduced lipofuscinosis in several brain regions of Grn Ϫ / Ϫ mice. AAV-Grn also reduced microgliosis in brain regions distant from the injection site. AAV-expressed progranulin was only detected in neurons, not in microglia, indicating that the microglial activation in progranulin deficiency can be improved by targeting neurons and thus may be driven at least in part by neuronal dysfunction. Even areas with sparse transduction and almost undetectable progranulin showed improvement, indicating that low-level replacement may be sufficiently effective. The beneficial effects of AAV-Grn did not require progranulin binding to sortilin. Finally, we tested whether AAV-Grn improved lysosomal function. AAV-derived progranulin was deliv- ered to the lysosome, ameliorated the accumulation of LAMP-1 in Grn Ϫ / Ϫ mice, and corrected abnormal cathepsin D activity. These data shed light on progranulin biology and support progranulin-boosting therapies for NCL and FTD due to GRN mutations. Key words: cathepsin D; frontotemporal dementia; gene therapy; lysosome; neuronal ceroid lipofuscinosis; progranulin Significance Statement Heterozygous loss-of-function progranulin (GRN) mutations cause frontotemporal dementia (FTD) and homozygous mutations cause neuronal ceroid lipofuscinosis (NCL). Here, we address several mechanistic questions about the potential of progranulin gene therapy for these disorders. GRN mutation carriers with NCL or FTD exhibit lipofuscinosis and Grn Ϫ / Ϫ mouse models develop a similar pathology. AAV-mediated progranulin delivery reduced lipofuscinosis in Grn Ϫ / Ϫ mice even after the onset of pathology. AAV delivered progranulin only to neurons, not microglia, but improved microgliosis in several brain regions, indi- cating cross talk between neuronal and microglial pathology. Its beneficial effects were sortilin independent. AAV-derived pro- granulin was delivered to lysosomes and corrected lysosomal abnormalities. These data provide in vivo support for the efficacy of progranulin-boosting therapies for FTD and NCL. Introduction among the leading causes of dominantly inherited frontotempo- Loss-of-function mutations in progranulin (GRN) cause neuro- ral dementia (FTD) (Baker et al., 2006; Cruts et al., 2006; Gass et degenerative disease with a gene-dose effect. GRN mutations are al., 2006). These GRN mutations typically cause progranulin hap- Received Oct. 25, 2017; revised Dec. 27, 2017; accepted Jan. 20, 2018. advice regarding MHCII immunostaining; Alexandra Nicholson for technical advice regarding progranulin/sortilin Author contributions: A.E.A. and E.D.R. designed research; A.E.A., V.C.O., and D.E.U. performed research; A.E.A., coimmunoprecipitation; Robert Farese, Jr., for providing Grn Ϫ / Ϫ mice; Joachim Herz for providing progranulin V.C.O., D.E.U., and E.D.R. analyzed data; A.E.A. and E.D.R. wrote the paper. constructs;YasuoUchiyamaforprovidingSCMASantibody;andTaleneYacoubianandDavidStandaertforproviding This work was supported by the Consortium for FTD Research and the Bluefield Project to Cure FTD, a Ruth L. AAVexpressionconstructs.BehaviorexperimentswereperformedintheEvelynF.McKnightBrainInstituteBehavior Kirschstein National Research Service Award Fellowship (F32NS090678), the National Institute of Neurological Core Facility. Disorders and Stroke–National Institutes of Health (NIH Grant R01NS075487, P30NS47466), the National Institute The authors declare no competing financial interests. on Aging–NIH (K99AG056597), and the Civitan International Research Center. We thank James Black and Miriam Correspondence should be addressed to either Erik D. Roberson or Andrew E. Arrant, Center for Neurodegenera- Robersonforhelpwithmousebreedingandcolonymanagement;AshleyHarmsforhelpfuldiscussionandtechnical tionandExperimentalTherapeutics,Alzheimer’sDiseaseCenter,EvelynF.McKnightBrainInstitute,Departmentsof Ϫ Ϫ 2342 • J. Neurosci., February 28, 2018 • 38(9):2341–2358 Arrant et al. • AAV-Progranulin Improves Pathology in Grn / Mice loinsufficiency, with plasma progranulin levels reduced by Ͼ50% gene therapy might work. We infused an AAV vector expressing relative to controls (Finch et al., 2009). Individuals with muta- mouse progranulin (AAV-Grn) or an AAV-GFP control virus tions in both GRN alleles, resulting in nearly complete progranu- into the medial prefrontal cortex (mPFC) of wild-type or lin deficiency, develop the lysosomal storage disorder neuronal Grn Ϫ / Ϫ mice after the onset of pathology and collected brain ceroid lipofuscinosis (NCL) (Smith et al., 2012; Canafoglia et al., tissue for analysis 8–10 weeks later. 2014; Almeida et al., 2016). NCL due to GRN mutations has been termed CLN11-NCL and is characterized by seizures and retinal Materials and Methods degeneration with onset in the early twenties (Smith et al., 2012; Animals. The line of progranulin-deficient mice used for this study was Canafoglia et al., 2014; Almeida et al., 2016). Despite very differ- generated and crossed onto a C57BL/6J background as described previ- ent clinical presentations, FTD patients with GRN mutations ously (Martens et al., 2012; Filiano et al., 2013). The mice used for this exhibit some pathological similarities to CLN11-NCL because study were obtained by breeding Grn ϩ/ Ϫ mice to produce wild-type, brains of patients with both diseases exhibit gliosis, increased Grn ϩ/ Ϫ, and Grn Ϫ / Ϫ mice. The resulting wild-type and Grn Ϫ / Ϫ litter- levels of lysosomal proteins, and lipofuscinosis (Go¨tzl et al., 2014; mates were used for all experiments. Male and female mice were included Ward et al., 2017). in the study. The mice were bred and housed in a barrier facility accred- Progranulin is a widely expressed, secreted glycoprotein that ited by the Association for Assessment and Accreditation of Laboratory performs a variety of functions (Bateman and Bennett, 1998; Animal Care. Mice were maintained on a 12 h:12 h light/dark cycle with Eriksen and Mackenzie, 2008; Cenik et al., 2012; Nguyen et al., lights on at 6:00 A.M. and were given ad libitum access to food (NIH-31 2013). Progranulin acts as a trophic factor for many cell types, diet #7917; Envigo) and water in all phases of the study. All experiments were approved by the Institutional Animal Care and Use Committee of including neurons (Van Damme et al., 2008; Ryan et al., 2009; the University of Alabama at Birmingham. Gass et al., 2012; Beel et al., 2017). It also modulates inflammation Antibodies. The following antibodies were used for immunostaining and facilitates wound healing (Zhu et al., 2002; Yin et al., 2010). and Western blot: CD68 (1:500 rat monoclonal, #MCA1957; Bio-Rad), The development of NCL in patients with complete progranulin Iba1 (1:500 rabbit polyclonal, #019-19741; Wako), MHCII (1:100 rat deficiency shows that progranulin is critical for proper lysosomal monoclonal, #14-5321-81; Thermo Fisher Scientific), GFAP (1:5000 rab- function (Smith et al., 2012). Recent data have shown that pro- bit polyclonal, #Z0334; Dako), NeuN (1:1000 mouse monoclonal, granulin is critical for proper trafficking and function of lyso- #MAB377; Millipore/Sigma-Aldrich), progranulin (1:500 for Western somal enzymes such as ␤-glucocerebrosidase and cathepsin D blot and chromogenic immunostaining, 1:200 for fluorescent immuno- (CatD), providing a potential mechanism by which progranulin staining, sheep polyclonal, #AF2557; R&D Systems), GFP (1: 1000 rabbit promotes lysosomal function (Jian et al., 2016; Beel et al., 2017; monoclonal, #2956; Cell Signaling Technology), CatD (1:500 goat poly- clonal, #sc-6486; Santa Cruz Biotechnology), LAMP-1 (1:250 rat mono- Valdez et al., 2017; Zhou et al., 2017b). Progranulin knock-out clonal, #1D4B; Developmental Studies Hybridoma Bank), sortilin (1:1000 mice have been used to study the effects of progranulin insuffi- rabbit polyclonal, #ab16640; Abcam), ␣-tubulin (1:1000 mouse monoclo- Ϫ / Ϫ ciency and model the gene-dose effect seen in humans. Grn nal, #T5168; Sigma-Aldrich), and SCMAS (1:300 rabbit polyclonal, pro- ϩ/ Ϫ mice, but not Grn mice, develop NCL-like pathology, includ- vided by Dr. Yasuo Uchiyama, Juntendo University) (Koike et al., 2000).